The major object of this proposal is to use genetic and biochemical methods to study the unique mechanism of RNA replication and transcription in coronaviruses. Coronaviruses are a family of single-stranded, positive-sense RNA viruses whose helical nucleocapsids are packaged within host-derived membrane envelopes containing a small complement of viral membrane glycoproteins. The RNA genomes of coronaviruses are among the largest mature RNA molecules yet discovered, making their genetic manipulation unapproachable by the techniques that have been used with other RNA viruses. An alternative system, targeted RNA recombination, has been developed for the site-specific mutagenesis of all the structural genes of the prototype coronavirus mouse hepatitis virus (MHV). This method takes advantage of the high rate of RNA-RNA recombination in MHV by transducing site-specific mutations into the viral genome by recombination with a synthetic RNA introduced into infected cells. Single point mutations and extensive substitutions have been engineered into MHV in this fashion. This procedure will now be used to study the roles played by genomic elements in viral RNA synthesis. First, the structure and mode of action of functional elements in the 3' untranslated region of the viral genome will be explored to learn how they participate in replication. Second, the roles of the sequence and the context of intergenic elements will be examined to gain an understanding of how viral messenger RNAs are produced. Coronaviruses are important respiratory, neurologic and enteric pathogens, and an understanding of their molecular biology is critical for their control and prophylaxis. The studies proposed will provide insights into the coronavirus life cycle and assembly, potential targets for antiviral chemotherapy, and a possible means to manipulate these infectious agents for vaccine design.